1. Field of the Invention
The present invention relates to the field of gene expression. More specifically, the invention relates to a system and method for regulating gene expression.
2. Description of the Prior Art
The bacterial .beta.-galactosidase gene (lacZ) is an excellent reporter gene and is used extensively in life science applications including: cloning (Sambrook et al., 1989. Molecular Cloning: A Laboratory Manual, 2.sup.nd ed. Cold Spring Harbor Laboratory Press, Plainview, N.Y.); promoter assessment (Park et al., 1994. A .beta.-galactosidase Expression vector for promoter analysis. DNA and Cell Biology. 13:1147-1149.); gene regulation and function; and mutation analysis (Gossen et al. 1989. Efficient rescue of integrated shuttle vectors from transgenic mice: a model for studying mutations in vivo. Proc. Natl. Acad. Sci. 86: 7981-7985). lacZ provides straightforward results regarding expression and function upon chromogenic assays using the substrate X-gal or ONPG; the quantitation of the protein is easily and reliably attained upon cell lysis, staining and spectrophotometric analysis; and the detection of in-vivo expression of the lacZ in mammalian cells is easily accomplished using basic histochemical staining.
It is of interest to have a reporter whereby transcription may be experimentally controlled. Although many eukaryotic promoters have been identified and cloned, they are often leaky (i.e., they do not provide total on-off control) and/or lack responsiveness in mammalian cell lines. Moreover, the inducers are generally deleterious to the host cell or have significant drawbacks such as toxicity.
Many chimeric transcription factors have been developed in order to ameliorate these problems. Earlier attempts at producing chimeric transcription factors were based on using the glucocorticoid receptor (Baniahmad et al., 1993. Mechanisms of transcriptional activation by steroid hormone receptors. J Cell Biochem. 51: 151-6). More recently the bacterial lac and tet operons have been exploited. The lac based systems have been characterised as "leaky" whereas the tet-based binary systems have exhibited greater fidelity between protein and operator sequences. Additionally, the tet-based systems are inducible using tetracycline, a well known and well characterised compound.
Two tetracycline responsive binary systems have been previously described (Deuschle et al., 1995. Tetracycline-reversible silencing of eukaryotic promoters, Molecular and Cellular Biology. 15: 1907-1914; and Gossen and Bujard, 1992. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc. Natl. Acad. Sci. USA. 89: 5547-5551, the contents of both of which are incorporated herein by reference.). Both the Deuschle and the Gossen and Bujard systems utilize a cis regulatory sequence composed of a heptameric repeat of the tetracycline operator (tetO) 19 bp inverted repeat fused to the immediate early sequences of the cytomegaloviral enhancer (TetO7CMV). However, the chimeric transcription factors are quite different. The tetracycline-controlled transactivator (tTA) system of Gossen and Bujard is composed of the tetR protein fused to the transactivating carboxy terminus of the VP-16 protein. The tetRKRAB chimera of Deuschle consists of the highly conserved KRAB (Kruppel-associated box) domain of the Kox1 Zn-finger protein family fused to the tetracycline repressor protein (tetRKRAB).
Accordingly, the motifs differ in their kinetics and mechanism of protein-DNA associations. The tTA system is induced by the absence of tetracycline. In the uninduced state however basal levels of expression tend to be relatively high due to the CMV sequences. The tetRKRAB system makes use of the transcriptional silencing influence of the KRAB domain. This system maintains much lower levels of basal activity in the repressed state, and is induced by the addition of tetracycline. Nevertheless, to date, induction levels are much lower than that observed in the tTA.
It is an object of the present invention to obviate and mitigate at least one of the disadvantages of conventional gene expression regulatory systems.